SUPPLEMENTARY INFORMATION FILE

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1 SUPPLEMENTARY INFORMATION FILE Existence of a microrna pathway in anucleate platelets Patricia Landry, Isabelle Plante, Dominique L. Ouellet, Marjorie P. Perron, Guy Rousseau & Patrick Provost 1. SUPPLEMENTARY FIGURES AND LEGENDS Supplementary Figure 1. Human platelets contain additional, unpublished mirna (mirplus) sequences. Equal amounts of RNA from purified platelet preparations, obtained from four different human donors, were pooled and subjected to mirna profiling analysis. The RNA samples were labeled using the mircury Hy3 /Hy5 labeling kit and hybridized on the mircury LNA Array (v.8.1) (Exiqon). For each mirplus capture probe, the mean signal from 4 replicate spots were averaged. The calculated standard error of the mean (SEM) is very low and is not visible on this plot. MiRNA detection was considered as positive when the calculated relative fluorescence intensity (RFI) for a given mirna capture probe was above the detection threshold. This threshold was conservatively set at 2 times the highest mean background signal observed in the array experiment. The data from each mirplus capture probe were plotted in the graph by order of intensity. mirplus micrornas are proprietary sequences that have yet to be annotated in mirbase.

2 Supplementary Figure 2. Human platelets contain a large amount of mirnas, as compared to human neutrophils and megakaryocytes. A subset (94) of the total number of mirnas analyzed by the mircury array were found to be differentially detected in platelets. The heat map diagram shows the result of the two-way hierarchical clustering of genes and samples. The clustering is performed on log2(hy3/hy5) ratios which passed the filtering criteria on variation across samples; standard deviation > 1.0 and > 0.5, respectively.

3 Supplementary Figure 3. The microrna profile of human platelets differ from that of neutrophils and megakaryocytes. Equal amounts of RNA from purified platelet and neutrophil (provided by the laboratory of Dr. Pierre Borgeat, Université Laval) preparations, obtained from four different human donors, and from megakaryocytes were pooled and subjected to mirna profiling analysis. The RNA samples were labeled using the mircury Hy3 /Hy5 labeling kit and hybridized on the mircury LNA Array (v.8.1) (Exiqon). For each mirna capture probe, the mean signal from 4 replicate spots were averaged. The calculated SEM is very low and is not visible in these bar graphs. MiRNA detection was considered as positive when the calculated relative fluorescence intensity (RFI) for a given mirna capture probe was above the detection threshold. This threshold was conservatively set at 2 times the highest mean background signal observed in the array experiment. More than 170 mirna capture probes gave a positive hybridization signal. The data from each of these probes were plotted in the bar graphs by order of intensity for platelets (upper panel) and aligned with the intensity of the corresponding mirna capture probe detected in neutrophils (center panel) and megakaryocytes (lower panel).

4 Supplementary Figure 4. Intraplatelet localization of the protein components of the mirna pathway. (a,b) Confocal and epifluorescence microscopy was performed on human platelets adherent on fibrinogen-coated glass coverslips (left) and megakaryocytes transferred onto microscope glass slides by cytospin (right), respectively, by adapting our published methodologies 6,47. (a) The presence of at least one representative protein of each of the catalytic steps of RNA silencing, i.e. DGCR8 (nuclear microprocessor complex), Dicer (pre-mirna processing complex) and Ago2 (RISC or mirnp effector complex), was assessed by using the following primary antibodies in combination with their related anti-mouse (dilution 1/500) or antirabbit (dilution 1/500) IgG secondary antibodies coupled with AlexaFluor488 (Molecular probe): anti-dgcr8 (Protein Tech Group, Inc, dilution 1/50), anti-dicer (dilution 1/50) 6 and anti-ago2 (Abnova, dilution 1/50) antibodies. (b, next page) The presence of α-tubulin was monitored by using an anti-tubulin antibody (anti-tat1 antibody 48, dilution 1/100) (a kind gift from Keith Gull, University of Oxford) in combination with the anti-mouse IgG secondary antibody, whereas the AlexaFluor488-labeled anti-mouse and anti-rabbit IgG secondary antibodies, incubated in the absence of primary antibodies, were used as controls. Platelet labeling was visualized with an inverted Olympus IX70 microscope (150X magnification), whereas staining in megakaryocytes was viewed with an Olympus BX51 microscope at 40X magnification. Images were prepared with Image J 1.38x software. Scale bar = 20 µm.

5 Supplementary Figure 4. Intraplatelet localization of the protein components of the mirna pathway. (cont d)

6 Supplementary Figure 5. Megakaryocytes can synthesize mirnas from pre-mirnas. (a) Megakaryocytes are capable of active mirna biosynthesis. Dicer processing activity in cleared protein extracts (10,000 g x 15 min supernatant; S10) and Dicer immunoprecipitates (IP) prepared from megakaryocytes was assessed upon incubation with a 32 P-labeled human let-7a-3 pre-mirna 30. (b) Characterization of a Dicer complex in megakaryocytes. Extracts from megakaryocytes were separated by gel filtration on a Superose 6 column and the fractions analyzed by Western blot using an anti-dicer antibody 6. Selected (odd) fractions were tested for their intrinsic Dicer activity upon addition of a 32 P-labeled human let-7a-3 pre-mirna substrate 30. (c) A Dicer TRBP2 complex is active in mirna biosynthesis in megakaryocytes. Immunoblot analysis of TRBP2 IP derived from platelet protein extracts by using an anti-dicer antibody (upper panel). Dicer processing activity in IP prepared from megakaryocytes by using anti-trbp2, anti-dicer or control rabbit IgG was assessed upon incubation with a 32 P-labeled human let-7a-3 pre-mirna 30 (lower panel). The reactions were analyzed by denaturing PAGE and autoradiography, as described 30. A 10-nt RNA ladder was used as a size marker. * indicates the expected mirna product.

7 Supplementary Figure 6. Megakaryocytes are functionally competent in RNA silencing. In vitro RISC activity assays performed using megakaryocyte protein extracts incubated in the presence of a 32 P-labeled RNA sensor bearing a binding site complementary to mir-223. The reactions were analyzed by denaturing PAGE and autoradiography. A 10-nt RNA ladder was used as a size marker (M).

8 Supplementary Figure 7. Characterization of an Ago2-containing effector complex competent in RNA silencing in megakaryocytes. (a,b) Extracts from megakaryocytes were separated by gel filtration on a Superose 6 column and the fractions analyzed for the presence of Ago2 by immunoblot analysis (a) and of mir-223 by Northern blot (b). (c) RISC activity assays were performed by using a 32 P-labeled mir-233 sensor transcript. * Indicates the expected 38-nt cleavage products.

9 Supplementary Figure 8. Characterization of a Dicer complex active in mirna biosynthesis and of an Ago2-containing effector complex competent in RNA silencing in HeLa cells. (a) Extracts from HeLa cells were separated by gel filtration on a Superose 6 column and the fractions analyzed by immunoblot analysis for the presence of Dicer and Ago2. (b) Selected (odd) fractions were tested for their intrinsic Dicer activity upon addition of a 32 P- labeled human let-7a-3 pre-mirna substrate 30. The reactions were analyzed by denaturing PAGE and autoradiography. A 10-nt RNA ladder was used as a size marker. * indicates the expected mirna product. (c) The fractions were analyzed by Northern blot for the presence of let-7c (mir-223 is not detected in HeLa cells). (d) RISC activity assays were performed by using a 32 P-labeled let-7c sensor transcript. * Indicates the expected 38-nt cleavage products.

10 Supplementary Figure 9. Characterization of polyclonal peptide antibodies used in this study. (a) Characterization of the antibody raised against human TRBP2 protein. Rabbits were immunized against the following peptide sequences used in combination as antigens (Eurogentec): TRBP2-1, 230 LDARDGNEVEPDDDHF 245 and TRBP2-2, 267 SLRNSVGEKILSLRSC 282. TRBP2-2-specific immunoglobulins (IgGs) were purified from collected serum by using peptide affinity chromatography (Eurogentec). The suitability of the human TRBP2 antibody for immunoblotting (IB) applications was validated upon analysis of protein extracts derived from HEK293 cells in the presence of increasing concentrations of blocking TRBP2-2 peptide (upper panel), whereas its ability to immunoprecipitate human TRBP2 was verified in HEK293 cells transiently expressing a Flag-tagged TRBP2 protein (lower panel). (b) Validation of the anti-dicer antibody 6 for immunoprecipitation (IP) applications. Protein extracts and Dicer IP derived from HEK293 cells transiently expressing a Flag-tagged Dicer protein were analyzed by IB using an anti-flag antibody.

11 2. SUPPLEMENTARY TABLE Supplementary Table 1. Relative fluorescence intensities (RFI) resulting from hybridization of the microrna probes on the array Order of MicroRNA probe Relative increasing (Exiqon mircury LNA Array fluorescence intensity v.8.1) intensity 1 hsa-mir hsa-mir-485-3p hsa-mir hsa-mir-423-3p hsa-mir-193a-3p hsa-mir hsa-mir-299-3p hsa-mir-542-5p hsa-mir hsa-mir hsa-mir-127-3p hsa-mir-374a hsa-mir hsa-mir hsa-mir-129-3p hsa-mir-92b hsa-mir hsa-mir hsa-mir-296-5p hsa-mir-502-3p hsa-mir hsa-mir hsa-mir hsa-mir-491-5p hsa-mir-525-5p hsa-mir hsa-mir-148a hsa-mir-551a hsa-mir hsa-mir hsa-mir hsa-mir-129-5p hsa-mir-324-5p hsa-mir hsa-mir-200c hsa-mir hsa-mir-33a hsa-mir hsa-mir-409-3p hsa-mir-518d-5p-520c-5p-526a hsa-mir hsa-mir hsa-mir-512-5p hsa-mir-518a-5p hsa-mir hsa-mir hsa-mir

12 48 hsa-mir hsa-mir-125b hsa-mir-486-5p hsa-mir-301a hsa-mir-99b hsa-let-7e hsa-mir-516b hsa-mir hsa-mir hsa-mir-519b-5p-519c-5p hsa-mir hsa-mir hsa-mir hsa-mir-376c hsa-mir hsa-mir hsa-mir-29b hsa-mir-10a hsa-mir hsa-mir-590-5p hsa-mir-339-5p hsa-mir-330-3p hsa-mir hsa-mir-376a hsa-mir-551b hsa-mir hsa-mir-19b hsa-mir-199a-5p hsa-mir-376b hsa-mir-28-5p hsa-mir hsa-mir-140-5p hsa-mir hsa-mir hsa-mir-361-5p hsa-mir hsa-mir-374b hsa-mir-342-3p hsa-mir-331-3p hsa-let-7g hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir hsa-mir-628-3p hsa-mir hsa-let-7f hsa-mir hsa-mir hsa-mir-20b hsa-mir-338-3p hsa-mir hsa-mir hsa-mir-30d hsa-mir-15a 1102

13 104 hsa-mir-29c hsa-mir hsa-mir-487b hsa-mir hsa-mir hsa-mir-769-3p hsa-mir hsa-mir-125a-5p hsa-mir hsa-mir-27a hsa-mir hsa-mir hsa-mir-30e hsa-mir hsa-mir-130b hsa-mir-27b hsa-mir hsa-mir hsa-mir hsa-mir-18b hsa-mir hsa-mir-30a hsa-mir-18a hsa-mir hsa-let-7d hsa-mir hsa-mir-148b hsa-mir hsa-mir-151-3p hsa-mir-146b-5p hsa-mir-106b hsa-mir-199a-3p-199b-3p hsa-mir hsa-mir hsa-mir-29a hsa-mir-671-5p hsa-mir hsa-mir hsa-mir-151-5p hsa-mir-20a hsa-mir hsa-mir hsa-mir hsa-mir-20a hsa-mir-15b hsa-mir-106a hsa-mir-19a hsa-mir-23a hsa-mir hsa-mir-23b hsa-mir-146a hsa-mir-30b hsa-mir hsa-mir-26a hsa-mir-130a hsa-mir-30c 14175

14 160 hsa-mir hsa-mir hsa-mir hsa-let-7b hsa-let-7i hsa-let-7c hsa-mir hsa-let-7a hsa-mir hsa-mir-142-3p hsa-mir-142-5p 47416

15 3. SUPPLEMENTARY REFERENCES 12. Provost, P. et al., Ribonuclease activity and RNA binding of recombinant human Dicer. Embo J 21 (21), (2002). 19. Ouellet, D.L. et al., Identification of functional micrornas released through asymmetrical processing of HIV-1 TAR element. Nucleic Acids Res 36 (7), (2008). 26. Provost, P. et al., Coactosin-like protein, a human F-actin-binding protein: critical role of lysine-75. Biochem J 359 (Pt 2), (2001). 27. Woods, A. et al., Definition of individual components within the cytoskeleton of Trypanosoma brucei by a library of monoclonal antibodies. J Cell Sci 93 ( Pt 3), (1989).